1. Field of the Invention
The present invention relates generally to the fields of molecular biology and biotechnology. More particularly, it concerns methods and compositions for isolating small RNA molecules that are typically 100 nucleotides or fewer, such as siRNA and miRNA, as opposed to larger RNA or DNA molecules. The isolated small RNA molecules can be used in subsequent studies or assays.
2. Description of Related Art
The study of small RNAs—RNA molecules on the order of 100 nucleotides or fewer—from various tissues in many organisms, as well as cultured cells, is an area of extreme interest now, and promises to remain one for the future. These small RNAs include microRNA molecules (miRNA) and small interfering RNA molecules (siRNA), both of which can have a powerful effect on the expression of a gene by virtue of hybridization to their target mRNA. Additionally, these procedures would be applicable to isolating small nuclear and small nucleolar RNAs (snRNAs and snoRNAs), involved in mRNA and rRNA processing. The procedures could also be used to isolate tRNAs along with 5S and 5.8S rRNAs, which are all involved in protein translation.
Key to these studies is the need to isolate RNA molecules in the size range of 15 to 100 nucleotides with high efficiency. Methods that provide a straightforward methodology to do this are therefore quite valuable.
The preparation of RNA from natural sources (tissue samples, whole organisms, cell cultures, bodily fluids) requires removal of all other biomolecules. Once water is eliminated, the primary component of cells is usually protein, often providing three-quarters of the mass. Of the major other biomolecules, lipids, carbohydrates, combinations of these with each other and protein, and DNA are the other main components. A goal of RNA extraction is to remove protein and DNA, as these provide the greatest interference in the use of RNA. Lipid and carbohydrate moieties can usually be dissolved away with the aid of a detergent. Protein can be stripped off RNA (and DNA) with the aid of detergents and denaturants, but still must be removed from the common solution.
Two main methods have historically been used to accomplish this end. The first is the use of organic solvents that are immiscible with water to dissolve (literally, to chemically extract) or precipitate proteins, after which the aqueous, protein-free phase can be separated by centrifugation prior to removal. Usually, phenol or phenol-chloroform mixtures are used for this purpose. The second method selectively immobilizes the RNA on a solid surface and rinses the protein away, after which conditions are used to release the RNA in an aqueous solution. This is literally a solid-phase extraction. Both procedures can reduce the amount of DNA contamination or carryover, with the efficiency varying with the precise conditions employed.
Phenol and phenol-chloroform extractions provide an extremely protein- and lipid-free solution of nucleic acid. Much if not all (depending on the sample) of the carbohydrate is also lost in this procedure as well. Acid phenol-chloroform is known to extract some of the DNA out of the aqueous solution (Chomczynski and Sacchi, 1987). However, the solution is high in denaturing agents such as guanidinium hydrochloride, guanidinium thiocyanate, or urea, all of which are incompatible with downstream enzymatic analysis, and the first two with electrophoretic analysis as well. RNA is usually separated from these mixtures by selective precipitation, usually with ethanol or isopropanol. This procedure is not as effective for small nucleic acid molecules, so this procedure is not ideal for the preparation of small RNAs.
Solid-phase extraction relies on high salt or salt and alcohol to decrease the affinity of RNA for water and increase it for the solid support used. The use of glass (silica) as a solid support has been shown to work for large RNAs in the presence of high concentrations of denaturing salts (U.S. Pat. Nos. 5,155,018; 5,990,302; 6,043,354; 6,110,363; 5,234,809; Boom et al., 1990) or lower concentrations of denaturing salts plus ethanol (U.S. Pat. No. 6,180,778). However, normal conditions for binding to glass fiber for RNA do not work for microRNA, and the use of a raw lysate is problematic due to variable requirements with different tissues.
Many of the protocols known involve isolation of DNA or larger mRNA, which are not ideal for isolation of small RNA molecules because these are often not effectively captured and eluted. Thus, there is a need for improved techniques for the efficient isolation, detection, and accurate quantification of these recently discovered small RNA molecules.